We studied changes in the expression of P-selectin on the blood platelet plasma membrane. The aim of the study was to determine the influence of renal carcinoma on P-selectin expression associated with changes in platelet morphology. Venous blood was collected from 30 patients with renal carcinoma and from 24 control subjects for cytometric analysis and to evaluate platelet morphology. P-selectin being the CD62P receptor on blood platelets was marked by anti-CD61/62P MoAb, and the results were presented as the percentage of CD62P-positive cells. Changes in the expression of the CD62P on the platelet plasma membrane during activation were investigated by flow cytometry in a comparative study of in vivo activation and in vitro platelet reactivity. Platelet activation reflected by P-selectin expression was higher in the group of patients (4.45 +/-1.96), compared to control (2.48 +/-1.66) (p < 0.05). However, adenosine diphosphate [ADP] -stimulated platelet reactivity in renal cancer patients increased only by 0.24% (p>> 0.05), while following activation by thrombin by 0.54% (p < 0.05). Moreover, a higher (4.72 +/-2.02), statistically significant percentage of platelets with P-selectin expression was found in patients with disseminated neoplastic changes in renal parenchyma, compared to patients with a single localized neoplastic lesion (4.17 +/-1.89) (p < 0.05). A statistically significant difference was noted in the platelet count and anisocytosis in renal cancer patients. Renal cancer enhances P-selectin expression. It is due to the presence of intensified thrombinogenesis and other platelet agonists in the blood.

OBJECTIVE

To investigate the effects of different aminoglycoside antibiotics on platelet aggregation and blood coagulation, as well as the underlying mechanisms.

METHODS

Blood samples were collected and prepared as platelet-rich plasma and platelet-poor plasma samples. Then assigned into different groups for the following antibiotics treatments: gentamicin, streptomycin, etimicin, amikacin, and kanamycin, as group 0 mg/L, group 30 mg/L, group 91mg/L, and group 910 mg/L for each drugs. The maximum platelet aggregation rate induced by adenosine diphosphate, expression levels of CD62p and FIB-R, prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen were measured. The sodium citrate and sodium heparin were used in whole blood tests for the whole blood coagulation time as well as the Ca(2+) in blood plasma.

RESULTS

Amikacin and gentamicin could inhibit the aggregation of platelets, which contributed to the whole blood clotting disorder.

CONCLUSIONS

Amikacin and gentamicin might inhibit the platelet aggregation by blocking the activation and release of FIB-R or probably the inhibition of endogenous clotting factor as well. This effect was not dependent on calcium ions.

Adhesion molecules play a critical role in the adhesive interactions of multiple cell types in sickle cell disease (SCD). We previously showed that anti-P-selectin aptamer efficiently inhibits cell adhesion to endothelial cells (ECs) and permits SCD mice to survive hypoxic stress. In an effort to discover new mechanisms with which to inhibit P-selectin, we examined the role of glycosylation. P-selectin is a 90 kDa protein but was found to migrate as 90 and 140 kDa bands on gel electrophoresis. When P-selectin isolated from ECs was digested with peptide N-glycosidase F, but not O-glycosidase, the 140 kDa band was lost and the 90 kDa band was enhanced. Treatment of ECs with tunicamycin, an N-glycosylation inhibitor, suppressed CD62P (P-selectin) expression on the cell surface as well as the 140 kDa form in the cytoplasm. These results indicate that the 140 kDa band is N-glycosylated and glycosylation is critical for cell surface expression of P-selectin in ECs. Thrombin, which stimulates P-selectin expression on ECs, induced AKT phosphorylation, whereas tunicamycin inhibited AKT phosphorylation, suggesting that AKT signaling is involved in the tunicamycin-mediated inhibition of P-selectin expression. Importantly, the adhesion of sickle red blood cells (sRBCs) and leukocytes to ECs induced by thrombin or hypoxia was markedly inhibited by two structurally distinct glycosylation inhibitors; the levels of which were comparable to that of a P-selectin monoclonal antibody which most strongly inhibited cell adhesion in vivo. Knockdown studies of P-selectin using short-hairpin RNAs in ECs suppressed sRBC adhesion, indicating a legitimate role for P-selectin in sRBC adhesion. Together, these results demonstrate that P-selectin expression on ECs is regulated in part by glycosylation mechanisms and that glycosylation inhibitors efficiently reduce the adhesion of sRBCs and leukocytes to ECs. Glycosylation inhibitors may lead to a novel therapy which inhibits cell adhesion in SCD.

We determined platelet-leucocyte complexes, which play roles in the thrombosis-inflammation relationship, in Behçet's disease patients with and without major vascular involvement (MVI) and in healthy controls. We included 36 Behçet's disease patients (22 male, 14 female, mean age: 34.4 +/- 8.3 years) and 20 healthy individuals (14 male, six female, mean age: 31.8 +/- 4.4 years). Whole blood count, CRP and ESR were determined in both groups. Clinical data about the patients were obtained from medical charts. Individuals with hypertension, diabetes, coronary artery disease, and smokers were excluded. Behçet's disease patients with MVI were taken as a separate group (8 male, 5 female, mean age: 37 +/- 8 years). MVI was defined as the presence of pulmonary arterial aneurysm, deep venous thrombosis, vena cava inferior or superior thrombosis, or venous sinus thrombosis. Flow cytometry was used to determine platelet-monocyte complexes (PMC), platelet-neutrophil complexes (PNC), basal and adenosine diphosphate (ADP)-stimulated platelet CD62P expression. Behçet's disease patients with MVI had significantly higher PNC than Behçet's disease patients without MVI and healthy controls (P values = 0.01). PMC levels in Behçet's disease patients with MVI were significantly higher than in healthy controls (P = 0.01). The groups were similar in basal and ADP-stimulated platelet CD62P expression (P values >0.05). Basal and ADP-stimulated CD62P expression, PMC and PNC were not significantly different between active Behçet's disease versus inactive Behçet's disease patients. The evaluated parameters were similar in Behçet's disease patients with and without uveitis, and pathergy-positive and pathergy-negative groups. Our results might suggest that the formation of PMC and PNC might play a role in thrombosis and MVI of Behçet's disease.

Circulating microparticles (cMPs) are small phospholipid-rich microvesicles shed by activated cells that play a pivotal role in cell signalling related to the pathogenesis of atherothrombosis. We aimed to investigate the prognostic value of cMPs released from different vascular cells for cardiovascular event (CVE) presentation in asymptomatic patients at high cardiovascular risk factors under nutritional and pharmacologic treatment. This is a nested case-control study of 50 patients from the five-year follow-up prospective PREDIMED trial enrolled in the nuts arm of the Mediterranean diet (MedDiet-nuts). We randomly selected 25 patients who had suffered a CVE during follow-up and pair-matched them for sex, age, and classical CV risk factors to 25 patients who remained asymptomatic (no-CVE). Total Annexin V-(AV)+ cMPs and cMPs from cells of the vascular compartment were quantified by flow cytometry at baseline and after one year follow-up. MedDiet-nuts and pharmacological treatment neither modified levels nor source of MP shedding in CVE patients. However, no-CVE patients showed 40-86 % decreased total AV+, PAC-1+/AV+, CD61+/AV+, CD142+/CD61+/AV+, CD62P+/AV+, CD146+/AV+, CD63+/AV+ and CD11a+/AV+ cMPs at one year follow-up (p≤0.046, all). CD142+/CD61+/AV+, CD146+/AV+ and CD45+/AV+ cMPs were decreased in no-CVE patients compared to CVE patients. A ROC-curve clustered model for CD142+/CD61+/AV+, CD45+/AV+ and CD146+/AV+ cMPs predicted a future CVE [p<0.0001, AUC=0.805 (0.672 to 0.938)]. In patients at high CV risk profile treated with a controlled MedDiet supplemented with nuts and receiving up-to-date CV drug treatment, reduced cMPs derived from activated platelets, leukocytes and endothelial cells are predictive of protection against CVE within the next four years.

OBJECTIVE

To investigate the significance of platelet-derived microparticles (PMPs) in women with recurrent spontaneous abortion.

METHODS

We measured platelet P-selectin (CD62P) as a platelet activation marker and CD42b(+) microparticles as PMPs by flow cytometry in whole blood of 20 women with recurrent spontaneous abortion and 20 age-matched healthy controls with no previous history of spontaneous abortion.

RESULTS

PMP levels in women with recurrent spontaneous abortion were higher than in women in the control group (4.79+/-1.18% vs 3.06+/-0.92%; P<0.000). CD62P levels were not significantly higher in the study group compared with the control group (13.78+/-8.62% vs 10.78+/-7.35%; P>0.05).

CONCLUSIONS

Our findings suggest that PMPs may have a role in the pathogenesis of recurrent spontaneous abortion.

In order to assess platelet activation by flow cytometry and activation-dependent monoclonal antibodies, a minute volume(2.5 microliters) of citrated whole blood was mixed for 15 minutes with a cocktail of monoclonal antibodies(MoAb), including PAC1, (a MoAb specific for fibrinogen receptors), and a MoAb against CD62P, (an alpha-granule membrane protein which associates with platelet surface membranes when platelets are activated). After fixation with 1% formaldehyde, the percentage of platelets positive for PAC1 and/or MoAb-CD62P was measured by flow cytometry. Even in resting platelets without stimulation, about 15% of platelets were found to be PAC1-positive and 0.6% of platelets were CD62P-positive. If there was a one-minute delay after needle puncture and before blood collection, the number of PAC1-positive platelets was increased compared to that of blood obtained immediately after puncture. If blood was allowed to stand at room temperature, there was a gradual increase in the number of PAC1-positive platelets, and after 60 minutes, this increase became statistically significant. The addition of Iloprost, (a prostacyclin analogue), immediately after venipuncture did not completely prevent the increase in PAC1-positive platelets after 60 minutes. If the inhibitor was added after first incubating for 60 minutes, however, the percentage of PAC1-positive platelets was reduced to preincubation value. Although flow cytometry is a simple and powerful tool to assess platelet activation, there remain several methodological problems to be resolved before this method may be employed in routine clinical use.

BACKGROUND

The aim of this study was to evaluate effects of Shen-Cao granules for the prevention of thrombocytopenia caused by anticancer chemotherapy.

METHODS

In this prospective study, a total of 200 patients with various malignant tumors were enrolled and evenly divided into a Shen-Cao granule treatment (n = 100) and a control group (n = 100). After 2 cycles chemotherapy with any combination of platinum-based drugs (cisplatin, carboplatin, and nedaplatin), the blood platelet (PLT) counts, levels of the PLT production regulator thrombopoietin (TPO), PLT aggregation rates, and the PLT activation marker CD62P expressions were monitored for 2 weeks.

RESULTS

During 2 weeks of post-chemotherapy, the mean values of the minimum PLT count were 49.65 ± 7.35 × 10/L in the treatment group and 31.56 ± 9.32 × 10/L in the control group. The PLT count in the treatment group reached the lowest value 1.8 days later and recovered to a concentration ≥100 × 10/L 3 days earlier than in the control group. The concentrations of the TPO were 71.43 ± 1.74 and 87.24 ± 0.92 ng/mL in the treatment group and 65.75 ± 1.39 and 67.75 ± 0.67 ng/mL in the control group at 7 and 14 days post-chemotherapy, respectively. The maximum PLT aggregation rate declined after chemotherapy in the treatment group from 58.14 ± 11.46% to 52.89 ± 10.52%, while it increased in the control group from 56.94 ± 10.55% to 61.75 ± 12.26%. Coordinately, the expression of CD62P in the treatment group decreased from 6.17 ± 0.59% to 4.89 ± 0.72%, while it increased from 6.09 ± 0.75% to 7.75 ± 0.67% in the control group.

CONCLUSIONS

Our study demonstrated that Shen-Cao granule treatment alleviated thrombocytopenia after chemotherapy, and reduced tumor-induced PLT activation and aggregation.

BACKGROUND

Hepatitis C virus (HCV) and Schistosoma mansoni are major causes of chronic liver disease (CLD) in which immune alteration is common. Recent studies suggested that certain platelets and lymphocytes activation markers may have an impact on progression of CLD. This study aimed to evaluate the potential of platelets and lymphocytes activation molecules expression on the pathogenesis of CLD in distinct or concomitant chronic HCV and schistosomiasis mansoni infections.

METHODS

The study populations were divided into group-I: patients with chronic schistosomiasis mansoni, group-II: HCV patients without cirrhosis, group-III: patients with combined liver diseases without cirrhosis, group-IV: patients with chronic HCV and liver cirrhosis and group-V: Age and sex matched healthy individuals as normal controls. All groups were subjected to full clinical evaluation, ELISA anti-HCV antibodies screening, parasitological examination for diagnosing S. mansoni and flow cytometry for lymphocyte (CD3, CD4, CD8, CD19, CD22, & CD56) and platelets activation (CD41, CD42 & CD62P (P- selectins)) markers.

RESULTS

The platelet count was significantly decreased in HCV and/or S. mansoni patients. The total T-lymphocytes and T-helper cells were significantly reduced, while T-cytotoxics were increased. The patients possessed a significantly higher platelets activation marker; CD62P (P-selectins) and higher mean fluorescent intensity (MFI) positivity. There were considerable correlations between platelets count and both of CD62P and MFI.

CONCLUSIONS

Our Findings suggest an increased expression of certain platelets and lymphocytes activation markers in chronic HCV and S. mansoni induced CLD that may have a role in disease progression.

OBJECTIVE

The expression of P-selectin on the surface of platelets and platelet-leucocyte conjugate formation are considered to be an indicator of platelet activation in thrombotic and inflammatory disease. Midazolam is a widely used sedative and anaesthetic induction agent. It may inhibit platelet aggregation and suppress interleukin-6 and -8 response in human leucocytes, but any effect on the adhesion of activated platelets to leucocytes remains obscure. We have examined the influence of midazolam on adenosine diphosphate (ADP)-induced platelet surface P-selectin expression and platelet-leucocyte aggregation in whole blood.

METHODS

Human whole blood was stimulated with 2 x 10(-5)M ADP in the presence of midazolam (3 x 10(-4) to 3 x 10(-6)M). Samples were stained with a fluorochrome-conjugated CD62P and CD41a antibody for detecting human platelet P-selectin antigens. The leucocyte subpopulations were separately gated and platelet-leucocyte aggregates were defined as cells found positive for CD45 and CD62P. All samples were analysed and were electronically separated into specific cell types (platelets, neutrophils, monocytes and lymphocytes) according to their typical forward/side scattering by flow cytometry.

RESULTS

Midazolam significantly inhibited ADP-induced platelet P-selectin expression and attenuated platelet-leucocyte aggregation (mainly in neutrophils and monocytes) in a dose-dependent manner with a maximum inhibitory effect at 3 x 10(-4)M (P < 0.01).

CONCLUSIONS

This study demonstrated that midazolam decreases the ADP-induced expression of platelet surface P-selectin and platelet-leucocyte aggregation.

Enumerating and phenotyping of platelets, resting and activated, from whole blood is important for both the identification and verification of many disease states. Microvolume laser scanning cytometry (MLSC) has been shown to be a simple method for enumerating and phenotyping peripheral blood cells. Here, the utility of MLSC, in conjunction with an anticoagulant containing platelet activation inhibitors, for simultaneously measuring platelet count, phenotype and responsiveness directly from non-fixed whole blood was examined. CTAD or EDTA anticoagulated blood was collected from five to 20 healthy volunteers, stained with fluorescence-labeled antibodies specific for platelet antigens, and run on an in-house modified MSLC device. MLSC was able to measure antigens CD9, CD29, CD36, CD41, CD42a, CD42b, and CD61 on platelets and determine an average of 2.3 x 10(5) +/- 7 x 10(4) platelets per microliter. Counts correlated well with those obtained from the Cell-Dyn 3500 (r(2)=0.84). Agreeing with previous data, less than 2% of platelets from peripheral blood of normal individuals expressed the activation markers CD62P or CD63. After in vitro thrombin activation, >93% of the platelets expressed activation markers. Data presented here shows the benefits of using MLSC in combination with platelet inhibitors to quantitate and phenotype platelets while maintaining a viable responsive state.

Millions of poor people in the developing world still thrive on ragpicking. In the present study, we have examined whether ragpicking is associated with increased risk of cardiovascular disease. For this, we have enrolled 112 premenopausal female ragpickers (median age 30 years) and 98 age-matched housemaids as control from Kolkata, Eastern India. Venous blood was drawn for routine hematology; flow cytometry was used to measure generation of reactive oxygen species (ROS) by leukocytes, surface expression of CD62P (P-selectin) in platelets and CD11b in leukocytes. Collagen-induced platelet aggregation was evaluated by aggregometer, and erythrocytic superoxide dismutase (SOD) was measured by spectrophotometry. Soluble P-selectin (sP-sel) and CD40L (sCD40L), neutrophil-activating protein-2 (NAP-2), platelet and plasma serotonin, oxidized low-density lipoprotein (oxLDL), and anticardiolipin antibodies (aCL) in plasma were measured by ELISA. Compared with control, the ragpickers had significantly higher prevalence of hypertension and prehypertension, and hypertension was positively associated with ragpicking. The ragpickers also had higher levels of inflammation (elevated NAP-2), oxidative stress (elevated ROS generation with depleted SOD) with oxLDL, platelet activation and aggregability, soluble CD40 ligand, with altered serotonin level (rose in plasma but depleted in platelet). A greater percentage of ragpickers had elevated serum level of aCL of the IgG and IgM isotypes than the controls. The results suggest that the occupation of ragpicking increases the risk of cardiovascular diseases in premenopausal women of Eastern India via inflammation, oxidative stress, platelet hyperactivity, and hypertension.

BACKGROUND

The Toll-like receptor 9 (TLR9) pathway can activate platelets but its role in acute coronary syndromes (ACS) is unknown. This study examined TLR9 expression and platelet activation in response to ODN2006, a TLR9 agonist, in healthy subjects and in ACS subjects treated with dual anti-platelet therapy (DAPT).

METHODS

TLR9 expression was examined in both resting and thrombin receptor activator peptide (TRAP)-activated platelets (1 and 10μM) from healthy and ACS subjects by flow cytometry. In both cohorts, ODN2006-mediated platelet activation (5μM) was examined in whole blood (WB) and platelet-rich plasma (PRP) using cell-surface CD62p and CD63 expression by flow cytometry.

RESULTS

Baseline TLR9 expression was significantly greater in ACS subjects compared to healthy subjects (p<0.01). Following TRAP activation, TLR9 expression increased dose-dependently in healthy subjects. However, no difference in TLR9 expression was seen in ACS platelets following TRAP activation. ODN2006 treatment resulted in significant increases in cell-surface expression of CD62p and CD63 in both WB (all p<0.001) and PRP (all p<0.001) in comparison to unstimulated platelets in healthy subjects. Despite DAPT, ODN2006 treatment produced significant increases in both activation markers in the ACS cohort across WB and PRP (all p<0.0001). Elevated baseline expression of TLR9 in ACS platelets may indicate increased sensitivity to TLR9 agonists and contribute to increased platelet activation in these patients. Furthermore, ODN2006 stimulation can activate platelets in ACS subjects despite treatment with DAPT.

CONCLUSIONS

This study demonstrates TLR9 expression and activation to be of potential therapeutic importance in ASC patients.

Background Acute vascular effects of high intensity physical activity are incompletely characterized. Circulating microparticles are cellular markers for vascular activation and damage. Methods Microparticles were analysed in 99 marathon runners (49 ± 6 years, 22% female) of the prospective Berlin Beat of Running study. Blood samples were taken within three days before, immediately after and within two days after the marathon run. Endothelial-derived microparticles were labelled with CD144, CD31 and CD62E, platelet-derived microparticles with CD62P and CD42b, leukocyte-derived microparticles with CD45 and monocyte-derived microparticles with CD14. Results Marathon running induced leukocytosis (5.9 ± 0.1 to 14.8 ± 0.3 109/l, p < 0.0001) and increased platelet counts (239 ± 4.6 to 281 ± 5.9 109/l, p < 0.0001) immediately after the marathon. Blood monocytes increased and lymphocytes decreased after the run ( p < 0.0001). Endothelial-derived microparticles were acutely increased ( p = 0.008) due to a 23% increase of apoptotic endothelial-derived microparticles ( p = 0.007) and returned to baseline within two days after the marathon. Thrombocyte-derived microparticles acutely increased by 38% accompanied by an increase in activated and apoptotic thrombocyte-derived microparticles ( p ≤ 0.0001) each. Both monocyte- and leukocyte-derived microparticles were decreased immediately after marathon run ( p < 0.0001) and remained below baseline until day 2. Troponin T increased from 12 to 32 ng/l ( p < 0.0001) immediately after the run and returned to baseline after two days. Conclusion Circulating apoptotic endothelial- and thrombocyte-derived microparticles increased after marathon running consistent with an acute pro-thrombotic and pro-inflammatory state. Exercise-induced vascular damage reflected by microparticles could indicate potential mechanisms of post-exertional cardiovascular complications. Further studies are warranted to investigate microparticles as markers to identify individuals prone to such complications.

BACKGROUND

Buffy-coat processing allows for the use of platelet additive solutions (PASs). PASs reduce plasma-associated transfusion reactions and conserve plasma for transfusion or fractionation. Platelet (PLT) storage in plasma was compared to storage in three commercially available PASs compared to assess their influence on in vitro laboratory variables.

METHODS

Platelet concentrates (PCs) were prepared from leukoreduced pools of four buffy coats (BCPs) suspended in autologous plasma or one of PASs (Composol, Fresenius-Kabi; T-Sol, Baxter Corp.; or SSP+, MacoPharma). On Days 1, 2, 3, 5, and 7 of storage, samples were tested for PLT concentration, mean PLT volume (MPV), CD62P, morphology, pO2, pCO2, glucose, lactate and total protein concentration, pH, extent of shape change (ESC), and hypotonic shock response (HSR). Data were analyzed by analysis of variance (ANOVA) with repeated measures and t tests.

RESULTS

PLT recoveries from BCPs were higher (p < 0.05) with plasma than any PAS. Storage medium and duration did not affect PLT concentration or MPV over time. CD62P expression and morphology were significantly different among PCs pooled with different media. ANOVA showed (p < 0.05) differences among the rates of change of pCO2, pH, glucose consumption, lactate production, and ESC; PASs such as Composol and SSP+ offered excellent maintenance of pH and low rates of glucose consumption. PAS performed poorly in ESC and HSR compared to plasma. Correlation studies reveal far more significant correlations between variables of PLTs in PAS than in plasma.

CONCLUSIONS

Newer PASs, for example, SSP+ and Composol, can maintain PLT integrity and moderate metabolism similarly to plasma but offer consistently lower PLT recoveries and limited osmotic balance.

Platelet (PLT) storage at cold temperatures (4°C) can reduce bacterial contamination and lower the risk of transfusion-related complications. We compared the effects of 22 and 4°C storage conditions for PLTs to further explore the efficiency of hemostasis in acute bleeding and extended PLT shelf life.

Manually prepared PLTs (PLT concentrates in plasma, not PLT additive solution) were stored at 4 and 22°C. The PLT counts, scanning electronic microscope observations, blood gas indices, biochemical indices, PLT aggregative function, and surface CD62P expression were monitored and compared between the groups.

There was no obvious change in PLT counts between Day 21 at 4°C and Day 5 at 22°C. PLTs stored at 4°C for 10 to 14 days were dramatically activated, had rough surfaces, and showed a significant degree of long pseudopodia formation. The pH of the PLTs on Day 5 was lower at 22°C than at 4°C, while the lactate dehydrogenase and lactic acid levels in the former group were significantly higher (p < 0.005). The maximum aggregation rates induced by collagen and arachidonic acid in the PLTs stored at 4°C for 5 days remained higher than 80%, while the rates induced by four inducers in the PLTs stored at 22°C were less than 5%. PLTs stored at 4°C for 10 to 14 days showed higher surface expression of PAC-1 and CD62P.

PLT counts, cellular morphologies, PLT membranes, cytoplasmic structures, aggregation rates, and hemostatic PLT function stored at 4°C for 10 to 14 days were better than those stored at 22°C for 5 days.

OBJECTIVE

Numerous studies have suggested that grape seed extract (GSE) confers vascular protection due to the direct effect of its polyphenol content on endothelial cells. The aim of the study was to determine whether GSE confers vascular protection through the direct effect of its polyphenol content on endothelial cells.

METHODS

After incubation with GSE-treated human umbilical vein endothelial cells (HUVECs), blood platelet reactivity was evaluated with regard to the expression of CD62P and the activated form of GPIIbIIIa in ADP-stimulated platelets.

RESULTS

Lower concentrations of GSE were found to enhance the antiplatelet action of HUVECs: 1 μg/ml GSE reduced platelet reactivity by about 10%. While platelet reactivity was not altered by HUVECs incubated with higher concentrations of GSE, HUVEC proliferation was significantly reduced by GSE of up to 10 μg gallic acid equivalent/ml.

CONCLUSIONS

The results of the study show that low doses of GSE potentiate the inhibitory action of HUVECs on platelet reactivity, which may account, at least partially, for the protective effects of grape products against cardiovascular diseases. In contrast, high concentrations of GSE significantly impair endothelial cell proliferation in vitro.

OBJECTIVE

The ADVIA 2120 Haematology Analyser is capable of measuring parameters that can be used as markers of platelet activation, mean platelet component (MPC), platelet component distribution width (PCDW) and mean platelet mass (MPM). This study investigated the degree of correlation of these measures of platelet granularity with CD62P measurement of platelet activation by flow cytometry in platelet concentrates.

METHODS

Pooled platelets in plasma/citrate phosphate dextrose (CPD) anticoagulant or apheresis platelets in plasma/acid citrate dextrose formula A (ACD-A) anticoagulant were evaluated. Pooled platelets were tested during 13 day storage, and apheresis platelets within 24 h of venepuncture. These were assessed for platelet activation using CD62P and the ADVIA, with or without extra EDTA anticoagulant.

RESULTS

In pooled platelets, PCDW correlated strongly with CD62P, both with and without the addition of extra EDTA anticoagulant. There was a good correlation between MPC and CD62P with additional EDTA, but a weaker correlation without extra EDTA. There was no correlation between CD62P and MPM. In apheresis platelets the correlation between PCDW and CD62P was poor, whereas MPC correlated strongly with CD62P if EDTA anticoagulant was added.

CONCLUSIONS

The usefulness of ADVIA platelet granularity measures to predict the degree of platelet activation depends upon the anticoagulant present in the platelet concentrate, and whether extra EDTA is added to the sample. Although ADVIA MPC and PCDW measurement could not replace CD62P or other gold standard methods of assessing platelet activation, these ADVIA 2120 parameters may provide a quick check of platelet concentrate quality.

Platelet-tumour cell interaction is implicated in the initiation of breast cancer-associated thrombosis, with hormone-therapy (Tamoxifen/Anastrozole), increasing this risk. However, recent in vitro research indicates that Tamoxifen inhibits platelet activation, while the effects of Anastrozole on platelet activation are not well characterised. This study investigated platelet activation caused by Tamoxifen or Anastrozole-treated breast cancer cells in vitro. MCF7 and T47D cells were pre-treated with Tamoxifen or Anastrozole to mimic the effects of the drugs in vivo, and co-cultured with whole blood. Platelet activation was determined using flow cytometry. Platelet (CD41a⁺CD62P⁺) was determined using an interval gating strategy. Platelet morphology was visualised using scanning electron microscopy. Our results support clinical findings, showing that hormone-therapy is associated with platelet activation. Tamoxifen-treated MCF7 cells increased P-selectin expression, with ultrastructural analysis showing fully spread platelets. Conversely, Tamoxifen-treated T47D cells decreased P-selectin expression with platelets showing signs of early aggregation. Anastrozole pre-treatment decreased P-selectin expression, with treated MCF7 cells inducing platelet membrane folds and lamellipodia extension, and treated T47D cells inducing platelet aggregation and fibrin network formation indicating hypercoagulation. The findings support clinical studies. Hormone-therapy augments tumour cell-induced platelet activation, which may be linked to cell phenotype. This may have clinical implications for treatment strategies.

BACKGROUND

Residual platelet reactivity is a predictor of poor prognosis in patients with acute coronary syndromes (ACSs) undergoing percutaneous coronary intervention. Thrombin is a major platelet activator and upon initiation of the coagulation cascade, it is subsequently produced downstream of factor IXa, which itself is known to be increased in ACS. Pegnivacogin is a novel RNA-aptamer based factor IXa inhibitor featuring a reversal agent, anivamersen. We hypothesized that pegnivacogin could reduce platelet reactivity.

METHODS

Whole blood samples from healthy volunteers were incubated in vitro in the presence and absence of pegnivacogin and platelet reactivity was analysed. In addition, platelet aggregometry was performed in blood samples from ACS patients in the RADAR trial featuring the intravenous administration of pegnivacogin as well as reversal by anivamersen.

RESULTS

In vitro, pegnivacogin significantly reduced adenosine diphosphate-induced CD62P-expression (100% vs. 89.79±4.04%, p=0.027, n=9) and PAC-1 binding (100% vs. 83.02±4.08%, p=0.010, n=11). Platelet aggregation was reduced (97.71±5.30% vs. 66.53±9.92%, p=0.013, n=10) as evaluated by light transmission aggregometry. In the presence of the RNA-aptamer reversal agent anivamersen, neither CD62P-expression nor platelet aggregation was attenuated. In patients with ACS treated with aspirin and clopidogrel, residual platelet aggregation was significantly reduced 20 min after intravenous bolus of 1 mg/kg pegnivacogin (100% versus 43.21±8.23%, p=0.020).

CONCLUSIONS

Inhibition of factor IXa by pegnivacogin decreases platelet activation and aggregation in vitro. This effect was negated by anivamersen. In ACS patients, platelet aggregation was significantly reduced after intravenous pegnivacogin. An aptamer-based anticoagulant inhibiting factor IXa therefore might be a promising antithrombotic strategy in ACS patients.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.